A solid-phase radioimmunoassay for ovine luteinizing hormone (LH) has been developed, utilizing antibody-coated polystyrene tubes for incubation of the assay and counting of the bound tracer. Tubes were coated with equine antiserum to bovine LH (Snook), and purified ovine LH (Papkoff) was used for iodination with 126 I and assay standards. The procedure is simple and rapid, being completely performed in the assay tubes and giving results after a period of 48 hr. Basal levels of plasma LH were 2.9 ±0.9 ng/ml in the cycling ewe, 1.2 ±0.9 ng/ml in the ram, 12.6 ±5.5 ng/ml in the oophorectomized ewe, and 1.2 ±0.9 ng/ml in the pregnant ewe. These values obtained from jugular venous plasma are 25% higher than those measured in peripheral venous plasma. The sheep does not appear to produce a placental gonadotrophin comparable to human chorionic gonadotrophin in regard to immunological crossreaction with pituitary LH. Plasma levels of LH rose sharply 4-16 hr after the onset of estrus to levels of 80-200 ng/ml, over a total period of only 10 hr. Frequent blood sampling is necessary to delineate the estrous LH release in the sheep, and daily estimations carry a greater than 50% chance of completely missing the LH peak. Administration of estradiol-17/3 to anestrous sheep by intramuscular injection and intravenous infusion was regularly followed by a typical estrous peak of LH secretion. The latent period of the estrogen stimulus was approximately 9 hr, and the dose required to produce an ovulatory LH peak was 6-10 ng, an amount similar to that secreted by the ovary at estrus. It is likely that estrogen secretion by the ovary is a major factor in stimulating the LH release accompanying estrus in the sheep (Endocrinology 85: 133, 1969) T HE STUDY of pituitary gonadotrophin secretion in the sheep was previously dependent upon indirect observations and the use of bioassay techniques. The timing of luteinizing hormone (LH) release has been examined by estimation of pituitary LH content (1) and by measurement of LH in blood obtained from the cavernous sinus (2). These studies have relied upon ovarian ascorbic acid depletion assays (3, 4) for quantitation of LH, a procedure which is adequate for the measurement of pituitary LH content but not generally regarded as satisfactory for the assay of blood LH levels. A decrease in pituitary LH content may indicate release of LH into the circulation, though this is uncertain unless the rate of hormone synthesis is also known, while cavernous sinus

1 Neurotransmitters released from nerve endings are inactivated by re-uptake into the presynaptic nerve terminals and possibly into neighbouring glial cells. While analysing the functional properties of a,-adrenoceptors in the hypothalamus, we observed a high-affinity uptake process for noradrenaline in postsynaptic peptidergic neurones.2 In primary hypothalamic cell cultures and in a hypothalamic neuronal cell line, [3H]-prazosin bound with high affinity and was displaced by unlabelled prazosin in concentrations of 10-10 to 10-7M. However, at concentrations of unlabelled prazosin above 10-7 M, there was a paradoxical increase in 9 The measured uptake of (-)-noradrenaline in the cell line was considerably increased by blockade of catechol-O-methyl-transferase and monoamine oxidase, suggesting that (-)-noradrenaline is metabolized to lipophilic products that escape across the plasma membrane. 10 Studies in rats, in which the noradrenaline isomer 6-hydroxydopamine was used, suggested that the postsynaptic uptake process is operative in hypothalamic CRH and vasopressin neurones in vivo.11 The Km for (-)-noradrenaline was within the range for the high affinity uptake, process in noradrenergic neurones. Uptake takes place in concentrations at which noradrenaline activates oxtadrenoceptors. Removal of noradrenaline from the vicinity of the receptors may prevent desensitization, thus maintaining the responsiveness of postsynaptic neurones to the actions of the neurotransmitter.

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Neurohypophysial hormones have been implicated in the control of anterior pituitary function, and oxytocin has been shown to stimulate gonadotrophin excretion and ovarian follicular development in certain species. To determine the role of neurohypophysial peptides in the control of gonadotrophin release, their actions on LH and FSH secretion were analysed in rats in vivo and in vitro. In adult female rats, administration of oxytocin during early pro-oestrus advanced the spontaneous LH surge and markedly increased peripheral LH levels at 15.00 h compared with control animals. In cultured pituitary cells from adult female rats, oxytocin and vasopressin elicited dose-related increases in LH and FSH release. Such responses were not affected by a potent gonadotrophin-releasing hormone (GnRH) antagonist that abolished GnRH agonist-induced release of LH and FSH. Oxytocin did not enhance GnRH agonist-stimulated gonadotrophin release to the same extent as it increased basal secretion, but at low concentrations of GnRH agonist the effects were additive. The gonadotrophin responses to oxytocin and vasopressin were inhibited by the specific neurohypophysial hormone antagonists, [d(CH2)5D-Ile2,Ile4,Arg8]vasopressin and [d(CH2)5Tyr (Me),Arg8]vasopressin. These results provide direct evidence that neurohypophysial hormones can stimulate gonadotrophin secretion through a receptor system distinct from the GnRH receptor. Such a mechanism could represent a complementary hypothalamic control system for long-term modulation of LH and FSH secretion by exerting a basal or tonic influence on gonadotrophin production.

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The effects of inhibition of phosphoinositide hydrolysis by U73122 [1-(6-[17 beta-3-methoxyestra-1,3,5- (10) triene-17-yl] amino/hexyl) 1H-pyrroledione] and neomycin on agonist-stimulated intracellular signaling and secretory responses were analyzed in cultured pituitary cells and alpha T3-1 gonadotrophs. GnRH (100 nM)- and endothelin-1 (ET-1; 100 nM)-induced inositol (1,4,5)-trisphosphate and diacylglycerol formation in normal cells and immortalized gonadotrophs were reduced by U73122 in a concentration-dependent manner, with IC50 values of about 2 microM and complete inhibition at 10 microM U73122. Neomycin also reduced GnRH- and ET-induced inositol phosphate production in both cell types. Agonist-induced intracellular Ca2+ responses were also inhibited in both cell types by U73122 and neomycin at the same concentrations that inhibited their inositol phosphate responses. In cultured pituitary cells, agonist-induced LH release was inhibited by U73122 and neomycin in a dose-dependent manner. In perifused pituitary cells, U73122 completely inhibited GnRH- and ET-1-induced LH release, but after 10 min caused a progressive and substantial increase in basal LH release. In static cultures, U73122 inhibited agonist-induced LH response at low concentrations (up to 3 microM), but stimulated LH release at higher concentrations due to direct activation of exocytosis by the compound. When added alone, U73122 caused a concentration-dependent increase in LH release with an EC50 of about 7 microM and a maximum response similar that that elicited by GnRH. The stimulatory action of U73122 on LH release was not reduced in the absence of extracellular Ca2+. In contrast to cultured pituitary cells, alpha T3-1 gonadotrophs showed only constitutive exocytosis that was not affected by either neomycin or U73122. These results demonstrate that GnRH and ET(A) receptors are coupled to the phosphoinositide/Ca2+ transduction system in pituitary gonadotrophs, and provide evidence for the dependence of agonist-regulated exocytosis on this signaling pathway. The ability of U73122 to stimulate LH release could reflect an additional action of the compound at late steps in the exocytic pathway.

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Twenty-day-old male rats were injected intraperitoneally with either human luteinizing hormone (HLH) or human growth hormone (HGH) labelled with 125I. The localization of these hormones 1\ p=n-\ 2 hr. after injection was examined under the light microscope after radioautography.Major sites of localization of labelled LH were the interstitial cells of the testis and the proximal convoluted tubule of the kidney. Some hormone was also present in adipose tissue, hepatic parenchymal cells, the mesothelial lining of the peritoneum and underlying macrophages. HGH was localized principally in the proximal convoluted tubule of the kidney with some hormone present in liver, adipose tissue, and the suprarenal cortex.